Modified landscapes can restrict the movement of organisms, leading to isolation and reduced population viability, particularly for species with extensive home ranges and long-distance travel, such as white-lipped peccaries (WLPs,Tayassu pecari). Previous studies have indicated that forested areas favor WLP herd movements, but the impact of the non-forested areas on their genetic connectivity is unknown. In this study, we used land cover, the Brazilian Roads Map, and population genetic data to investigate the impact of non-forested matrices on WLP’s genetic connectivity in the Pantanal floodplain and surrounding Cerrado plateau of central-west Brazil. We compared isolation-by-distance (IBD), isolation-by-barrier, and isolation-by-resistance models and tested 39 hypotheses within a modeling framework. Finally, we identified the optimal areas for ecological corridors based on the most effective landscape model. Barrier and landscape resistance were more strongly correlated with genetic relatedness than the IBD model. The model that received the most robust support considered only forest as habitat. All other classes formed a matrix that impeded gene flow, including agriculture, grassland, savannah, and paved and unpaved roads. WLP herds living in landscapes with reduced forest cover are more vulnerable to the effects of genetic isolation. To maintain gene flow, it is essential to establish connections between habitats throughout the landscape. Conservation programs should prioritize strategies that strengthen connections between habitats, including facilitating wildlife road-crossing structures and creating/restoring ecological corridors to link isolated habitat fragments.